P
US8962292B2ActiveUtilityPatentIndex 73

Capping-prone RNA polymerase enzymes and their applications

Assignee: JAIS PHILIPPEPriority: Apr 16, 2010Filed: Apr 15, 2011Granted: Feb 24, 2015
Est. expiryApr 16, 2030(~3.8 yrs left)· nominal 20-yr term from priority
Inventors:JAIS PHILIPPE
A61P 3/10A61P 35/00A61P 31/18A61P 7/04A61P 9/06A61P 31/16A61P 9/00A61P 31/14A61P 31/20A61P 37/06A61P 25/14A61P 25/18A61P 25/28A61P 25/16C12N 9/1241C12Y 306/04013C12Y 201/01056A61K 38/00C12N 9/14C07K 2319/00C12N 9/1007A61P 19/04A61P 1/16C12N 9/1247C12P 19/34A61P 17/00C12N 9/12A61P 1/00C12N 9/10C12N 15/79C12N 15/62Y02A50/30
73
PatentIndex Score
9
Cited by
23
References
34
Claims

Abstract

The invention provides a chimeric enzyme comprising at least one catalytic domain of a RNA triphosphatase, at least one catalytic domain of a guanylyltransferase, at least one catalytic domain of a N 7 -guanine methyltransferase, and at least one catalytic domain of a DNA-dependant RNA polymerase. The invention also provides pharmaceutical composition comprising said chimeric enzyme and uses of said chimeric enzyme.

Claims

exact text as granted — not AI-modified
The invention claimed is: 
     
       1. A hetero-oligomeric enzyme comprising:
 a first monomer and a second monomer linked together covalently or noncovalently, the first monomer comprising
 at least one catalytic domain of a RNA triphosphatase, 
 at least one catalytic domain of a guanylyltransferase, and 
 at least one catalytic domain of a N 7 -guanine methyltransferase, and the second monomer comprising 
 at least one catalytic domain of a DNA-dependent RNA polymerase, wherein the hetero-oligomeric enzyme is non-natural. 
 
 
     
     
       2. The hetero-oligomeric enzyme according to  claim 1 , wherein said catalytic domain of a DNA-dependent RNA polymerase is a catalytic domain of a bacteriophage DNA-dependent RNA polymerase. 
     
     
       3. The hetero-oligomeric enzyme according to  claim 1 , wherein at least one of
 said catalytic domain of a RNA triphosphatase; 
 said catalytic domain of a guanylyltransferase; and 
 said catalytic domain of a N 7 -guanine methyltransferase is a catalytic domain of a virus capping enzyme. 
 
     
     
       4. A mixture of isolated nucleic acid molecules, comprising a first nucleic acid molecule encoding the first monomer of the hetero-oligomeric enzyme according to  claim 1  and a second nucleic acid molecule encoding the second monomer of the hetero-oligomeric enzyme according to  claim 1 . 
     
     
       5. The mixture of isolated nucleic acid molecules according to  claim 4 , wherein the first nucleic acid molecule and/or the second nucleic acid molecule is/are operatively linked to at least one promoter selected from the group consisting of:
 a promoter for RNA polymerase II; and 
 a promoter for said catalytic domain of a DNA-dependent RNA polymerase. 
 
     
     
       6. A mixture of vectors encoding the hetero-oligomeric enzyme according to  claim 1 , comprising a first vector comprising a first nucleic acid molecule that encodes the first monomer of the hetero-oligomeric enzyme according to  claim 1  and a second vector comprising a second nucleic acid molecule that encodes the second monomer of the hetero-oligomeric enzyme according to  claim 1 . 
     
     
       7. A host cell comprising the mixture of isolated nucleic acid molecules according to  claim 4 . 
     
     
       8. An in vitro or ex vivo method for producing a RNA molecule with 5′-terminal m 7 GpppN cap encoded by a DNA sequence, in a host cell, said method comprising the step of expressing in the host cell the mixture of isolated nucleic acid molecules according to  claim 4 , wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependent RNA polymerase. 
     
     
       9. A kit for the production of a RNA molecule with 5′-terminal m 7 GpppN cap, comprising at least one hetero-oligomeric enzyme according to  claim 1 , and/or
 a mixture of isolated nucleic acid molecules, comprising a first nucleic acid molecule encoding the first monomer of the hetero-oligomeric enzyme according to  claim 1  and a second nucleic acid molecule encoding the second monomer of the hetero-oligomeric enzyme according to  claim 1 , and/or 
 a mixture of vectors encoding the hetero-oligomeric enzyme according to  claim 1 , comprising a first vector comprising a first nucleic acid molecule that encodes the first monomer of the hetero-oligomeric enzyme according to  claim 1  and a second vector comprising a second nucleic acid molecule that encodes the second monomer of the hetero-oligomeric enzyme according to  claim 1 . 
 
     
     
       10. A pharmaceutical composition comprising a hetero-oligomeric enzyme according to  claim 1 , and/or
 a mixture of isolated nucleic acid molecules, comprising a first nucleic acid molecule encoding the first monomer of the hetero-oligomeric enzyme according to  claim 1  and a second nucleic acid molecule encoding the second monomer of the hetero-oligomeric enzyme according to  claim 1 , and/or 
 a mixture of vectors encoding the hetero-oligomeric enzyme according to  claim 1  comprising a first vector comprising a first nucleic acid molecule that encodes the first monomer of the hetero-oligomeric enzyme according to  claim 1  and a second vector comprising a second nucleic acid molecule that encodes the second monomer of the hetero-oligomeric enzyme according to  claim 1 . 
 
     
     
       11. The pharmaceutical composition according to  claim 10 , further comprising:
 at least one DNA sequence of interest, wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependent RNA polymerase. 
 
     
     
       12. A hetero-oligomeric enzyme comprising:
 a dimer and a monomer linked together covalently or noncovalently, the dimer comprising:
 at least one catalytic domain of a RNA triphosphatase, 
 at least one catalytic domain of a guanylyltransferase, and 
 at least one catalytic domain of a N 7 -guanine methyltransferase, and the monomer comprising 
 at least one catalytic domain of a DNA-dependent RNA polymerase, wherein the hetero-oligomeric enzyme is non-natural. 
 
 
     
     
       13. The hetero-oligomeric enzyme according to  claim 12 , wherein said catalytic domain of a DNA-dependent RNA polymerase is a catalytic domain of a bacteriophage DNA-dependent RNA polymerase. 
     
     
       14. The hetero-oligomeric enzyme according to  claim 12 , wherein at least one of
 said catalytic domain of a RNA triphosphatase; 
 said catalytic domain of a guanylyltransferase; and 
 said catalytic domain of a N 7 -guanine methyltransferase 
 
       is a catalytic domain of a virus capping enzyme. 
     
     
       15. A mixture of isolated nucleic acid molecules, comprising a first nucleic acid molecule and a second nucleic acid molecule encoding the dimer of the hetero-oligomeric enzyme according to  claim 12  and a third nucleic acid molecule encoding the monomer of the hetero-oligomeric enzyme according to  claim 12 . 
     
     
       16. The mixture of isolated nucleic acid molecules according to  claim 15 , wherein any one of the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule is operatively linked to at least one promoter selected from the group consisting of:
 a promoter for RNA polymerase II; and 
 a promoter for said catalytic domain of a DNA-dependent RNA polymerase. 
 
     
     
       17. A mixture of vectors encoding the hetero-oligomeric enzyme according to  claim 12 , comprising a first vector comprising a first nucleic acid molecule and a second vector comprising a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule encoding the dimer of the hetero-oligomeric enzyme according to  claim 12 , and a third vector comprising a third nucleic acid molecule that encodes the monomer of the hetero-oligomeric enzyme according to  claim 12 . 
     
     
       18. A host cell comprising the mixture of isolated nucleic acid molecules according to  claim 15 . 
     
     
       19. An in vitro or ex vivo method for producing a RNA molecule with 5′-terminal m 7 GpppN cap encoded by a DNA sequence, in a host cell, said method comprising the step of expressing in the host cell the mixture of isolated nucleic acid molecules according to  claim 15 , wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependent RNA polymerase. 
     
     
       20. A kit for the production of a RNA molecule with 5′-terminal m 7 GpppN cap, comprising at least one hetero-oligomeric enzyme according to  claim 12 , and/or
 a mixture of isolated nucleic acid molecules, comprising a first nucleic acid molecule and a second nucleic acid molecule encoding the dimer of the hetero-oligomeric enzyme according to  claim 12  and a third nucleic acid molecule encoding the monomer of the hetero-oligomeric enzyme according to  claim 12 , and/or 
 a mixture of vectors encoding the hetero-oligomeric enzyme according to  claim 12 , comprising a first vector comprising a first nucleic acid molecule and a second vector comprising a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule encoding the dimer of the hetero-oligomeric enzyme according to  claim 12 , and a third vector comprising a third nucleic acid molecule that encodes the monomer of the hetero-oligomeric enzyme according to  claim 12 . 
 
     
     
       21. A pharmaceutical composition comprising a hetero-oligomeric enzyme according to  claim 12 , and/or
 a mixture of isolated nucleic acid molecules, comprising a first nucleic acid molecule and a second nucleic acid molecule encoding the dimer of the hetero-oligomeric enzyme according to  claim 12  and a third nucleic acid molecule encoding the monomer of the hetero-oligomeric enzyme according to  claim 12 , and/or 
 a mixture of vectors encoding the hetero-oligomeric enzyme according to  claim 12  comprising a first vector comprising a first nucleic acid molecule and a second vector comprising a second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule encoding the dimer of the hetero-oligomeric enzyme according to  claim 12 , and a third vector comprising a third nucleic acid molecule that encodes the—monomer of the hetero-oligomeric enzyme according to  claim 12 . 
 
     
     
       22. The pharmaceutical composition according to  claim 21 , further comprising:
 at least one DNA sequence of interest, wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependent RNA polymerase. 
 
     
     
       23. A monomeric enzyme comprising:
 at least one catalytic domain of a RNA triphosphatase, 
 at least one catalytic domain of a guanylyltransferase, 
 at least one catalytic domain of a N 7 -guanine methyltransferase, and 
 at least one catalytic domain of a DNA-dependent RNA polymerase, wherein the monomeric enzyme is non-natural. 
 
     
     
       24. The monomeric enzyme according to  claim 23 , wherein said catalytic domain of a DNA-dependent RNA polymerase is a catalytic domain of a bacteriophage DNA-dependent RNA polymerase. 
     
     
       25. The monomeric enzyme according to  claim 23 , wherein at least one of
 said catalytic domain of a RNA triphosphatase; 
 said catalytic domain of a guanylyltransferase; and 
 said catalytic domain of a N 7 -guanine methyltransferase is a catalytic domain of a virus capping enzyme. 
 
     
     
       26. The monomeric enzyme according to  claim 23 , wherein at least two of
 said catalytic domain of a RNA triphosphatase, 
 said catalytic domain of a guanylyltransferase, 
 said catalytic domain of a N 7 -guanine methyltransferase, and 
 said catalytic domain of a DNA-dependent RNA polymerase are bound by a linking peptide. 
 
     
     
       27. An isolated nucleic acid molecule encoding the monomeric enzyme according to  claim 23 . 
     
     
       28. The isolated nucleic acid molecule according to  claim 27 , which is operatively linked to at least one promoter selected from the group consisting of:
 a promoter for RNA polymerase II; and 
 a promoter for said catalytic domain of a DNA-dependent RNA polymerase. 
 
     
     
       29. A vector comprising the isolated nucleic acid molecule according to  claim 27 . 
     
     
       30. A host cell comprising the isolated nucleic acid molecule according to  claim 27 . 
     
     
       31. An in vitro or ex vivo method for producing a RNA molecule with 5′-terminal m 7 GpppN cap encoded by a DNA sequence, in a host cell, said method comprising the step of expressing in the host cell the isolated nucleic acid molecule according to  claim 27 , wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependent RNA polymerase. 
     
     
       32. A kit for the production of a RNA molecule with 5′-terminal m 7 GpppN cap, comprising at least one monomeric enzyme according to  claim 23 , and/or
 an isolated nucleic acid molecule encoding the monomeric enzyme according to  claim 23 , and/or 
 a vector comprising the isolated nucleic acid molecule encoding the monomeric enzyme according to  claim 23 . 
 
     
     
       33. A pharmaceutical composition comprising a monomeric enzyme according to  claim 23 , and/or
 an isolated nucleic acid molecule encoding the monomeric enzyme according to  claim 23 , and/or 
 a vector comprising the isolated nucleic acid molecule encoding the monomeric enzyme according to  claim 23 . 
 
     
     
       34. The pharmaceutical composition according to  claim 33 , further comprising:
 at least one DNA sequence of interest, wherein said DNA sequence is operatively linked to a promoter for said catalytic domain of a DNA-dependent RNA polymerase.

Cited by (0)

No later patents cite this yet.

References (0)

No backward citations on record.